An effort has been made to develop an in vitro system for the study of multistep oncogenic transformation of cells. A number of cell lines have been recently isolated by DNA-mediated transfection of the rat cell REF52 with cloned viral and cellular oncogenes. Lines expressing the SV40 T/t antigens or the activated ras oncogene (T24 H-ras 1) + adenovirus-5 early region 1A gene (Ad5E1A) are fully transformed, whereas lines expressing only the T24 H-ras 1 gene or the Ad5E1A gene are not. Numerous clones from each line have been characterized with respect to morphology, growth rate, serum dependence, oncogenicity, and expression of the p21 ras protein products as seen on 2D-gels. Initial results indicate that expression of the normal cellular p21 genes is altered as a result of expression of the T24 H-ras 1 gene, and that cells containing higher amounts of the T24 H-ras 1 gene products are less tumorigenic than cells containing lower amounts of the gene products. This research will further characterize these cell lines by computer-analyzed two-dimensional gel electrophoresis of cell cycle specific populations of cells, sorted as a function of DNA content. By studying cells at representative stages of the cell cycle we will determine: (1) detailed patterns of protein synthesis and turnover throughout the cell cycle; (2) the responses of each line to stimulation of growth factors after serum-deprivation; and (3) the changes that occur in each line during in vivo tumorigenesis. These experiments will show how basal levels of gene expression are affected by the presence of T24 H-ras 1 or E1A genes, or both, and they will show how the normal responses to serum and to purified growth stimulatory and inhibitory factors are altered each line. We will also continue studying the effect of introducing each of these genes into normal REF52 cells by microinjection of the cloned gene. Initial studies using 2D gel analysis of microinjected cells confirm the effect of T24 H-ras 1 gene expression on the detected steady-state levels of the cellular ras genes. (S)